Bioarray chip reaction apparatus and its manufacture

ABSTRACT

A body  300  having a cavity  310  for mounting a substrate  120  fabricated with probe sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, is provided. The cavity includes inlets  350  and  360  for introducing selected fluids into the cavity to contact the probes. Accordingly, a commercially feasible device for use in high throughput assay systems is provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/992,043, filed on Nov. 17, 2004, which is a continuation of U.S.patent application Ser. No. 10/619,224, filed Jul. 12, 2003, which is acontinuation of U.S. patent application Ser. No. 10/229,759, filed Aug.28, 2002, now U.S. Pat. No. 6,733,977, which is a continuation of U.S.patent application Ser. No. 10/046,623, filed Jan. 14, 2002, now U.S.Pat. No. 6,551,817, which is a continuation of U.S. patent applicationSer. No. 09/907,196, filed Jul. 17, 2001, now U.S. Pat. No. 6,399,365,which is a continuation of U.S. patent application Ser. No. 09/302,052,filed Apr. 29, 1999, now U.S. Pat. No. 6,287,850, which is acontinuation of U.S. patent application Ser. No. 08/485,452, filed Jun.7, 1995, now U.S. Pat. No. 5,945,334, which is a continuation-in-part ofU.S. patent application Ser. No. 08/255,682, filed Jun. 8, 1994, nowabandoned. Each of these applications is incorporated herein in itsentirety by reference for all purposes.

BACKGROUND OF INVENTION

The present inventions relate to the fabrication and placement ofmaterials at known locations on a substrate. In particular, oneembodiment of the invention provides a method and associated apparatusfor packaging a substrate having diverse sequences at known locations onits surface.

Techniques for forming sequences on a substrate are known. For example,the sequences may be formed according to the pioneering techniquesdisclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO 92/10092,or U.S. application Ser. No. 08/249,188, now U.S. Pat. No. 5,571,639,incorporated herein by reference for all purposes. The preparedsubstrates will have a wide range of applications. For example, thesubstrates may be used for understanding the structure-activityrelationship between different materials or determining the sequence ofan unknown material. The sequence of such unknown material may bedetermined by, for example, a process known as sequencing byhybridization. In one method of sequencing by hybridization, a sequencesof divers materials are formed at known locations on the surface of asubstrate. A solution containing one or more targets to be sequenced isapplied to the surface of the substrate. The targets will bind orhybridize with only complementary sequences on the substrate.

The locations at which hybridization occurs can be detected withappropriate detection systems by labeling the targets with a fluorescentdye, radioactive isotope, enzyme, or other marker. Exemplary systems aredescribed in U.S. Pat. No. 5,143,854 (Pirrung et al.) and U.S. patentapplication Ser. No. 08/143,312, also incorporated herein by referencefor all purposes. Information regarding target sequences can beextracted from the data obtained by such detection systems.

By combining various available technologies, such as photolithographyand fabrication techniques, substantial progress has been made in thefabrication and placement of diverse materials on a substrate. Forexample, thousands of different sequences may be fabricated on a singlesubstrate of about 1.28 cm² in only a small fraction of the timerequired by conventional methods. Such improvements make thesesubstrates practical for use in various applications, such as biomedicalresearch, clinical diagnostics, and other industrial markets, as well asthe emerging field of genomics, which focuses on determining therelationship between genetic sequences and human physiology.

As commercialization of such substrates becomes widespread, aneconomically feasible and high-throughput device and method forpackaging the substrates are desired.

SUMMARY OF THE INVENTION

Methods and devices for packaging a substrate having an array of probesfabricated on its surface are disclosed. In some embodiments, a bodycontaining a cavity is provided. A substrate having an array of probesis attached to the cavity using, for example, an adhesive. The bodyincludes inlets that allow fluids into and through the cavity. A seal isprovided for each inlet to retain the fluid within the cavity. Anopening is formed below the cavity to receive a temperature controllerfor controlling the temperature in the cavity. By forming a sealedthermostatically controlled chamber in which fluids can easily beintroduced, a practical medium for sequencing by hybridization isprovided.

In other embodiments, the body is formed by acoustically welding twopieces together. The concept of assembling the body from two pieces isadvantageous. For example, the various features of the package (i.e.,the channels, sealing means, and orientation means) are formed withoutrequiring complex machining or designing. Thus, the packages areproduced at a relatively low cost.

In connection with one aspect of the invention, a method for making thechip package is disclosed. In particular, the method comprises the stepsof first forming a plurality of probe arrays on a substrate andseparating the substrate into a plurality of chips. Typically, each chipcontains at least one probe array. A chip is then mated to a packagehaving a reaction chamber with fluid inlets. When mated, the probe arrayis in fluid communication with the reaction chamber.

In a specific embodiment, the present invention provides an apparatusfor packaging a substrate. The present apparatus includes a substratehaving a first surface and a second surface. The first surface includesa probe array and the second surface is an outer periphery of the firstsurface. The present apparatus also includes a body having a mountingsurface, an upper surface, and a cavity bounded by the mounting surfaceand the upper surface. The second surface is attached to the cavity andthe first surface is within the cavity. A cover attached to the mountingsurface for defining an upper boundary to the cavity is also included.The cavity includes a diffuser and a concentrator. The diffuser and theconcentrator permit laminar fluid flow through the cavity.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a illustrates a wafer fabricated with a plurality of probearrays.

FIG. 1 b illustrates a chip.

FIG. 2 a illustrates a scribe and break device.

FIG. 2 b illustrates the wafer mounted on a pick and place frame.

FIGS. 2 c-2 d illustrate the wafer, as displayed by the scribe and breakdevice during alignment.

FIG. 3 illustrates a chip packaging device.

FIG. 4 illustrates the chip packaging device assembled from twocomponents.

FIGS. 5 a-5 b illustrate the top and bottom view of a top casing of thechip packaging device.

FIG. 5 c illustrates a different cavity orientation.

FIG. 6 illustrates a cross sectional view of the packaging device.

FIG. 7 illustrates the bottom view of a bottom casing of the chippackaging device.

FIGS. 8 a-8 b illustrate an acoustic welding system.

FIGS. 9 a-9 c illustrate the acoustic welding process used in assemblingthe chip packaging device.

FIG. 10 illustrates an adhesive dispensing system used in attaching thechip to the chip packaging device.

FIGS. 11, 12 a, 12 b and 13 illustrate in greater detail the adhesivedispensing system of FIG. 10.

FIGS. 14 a-14 d illustrate the procedure for aligning the system of FIG.10.

FIGS. 15 a-15 e illustrate images obtained during the alignment p ofFIGS. 14 a-14 d.

FIGS. 16 a-16 b illustrate an alternative embodiment of a packagingdevice.

FIGS. 17 a-17 b illustrate another embodiment of a packaging device.

FIG. 18 illustrates an alternative embodiment for attaching the chip tothe packaging device.

FIG. 19 illustrates another embodiment for attaching the chip to thepackaging device.

FIGS. 20 a-20 b illustrate yet another embodiment for attaching the chipto the packaging device.

FIG. 21 illustrates an alternative embodiment for attaching the chip tothe packaging device.

FIG. 22 illustrates another embodiment for attaching the chip to thepackaging device.

FIG. 23 illustrates an alternative embodiment for sealing the cavity onthe packaging device.

FIG. 24 illustrates another alternative embodiment for sealing thecavity on the packaging device.

FIG. 25 illustrates yet another embodiment for sealing the cavity on thepackaging device.

FIGS. 26 a-26 b illustrate an alternative embodiment for sealing thecavity on the packaging device.

FIGS. 27 a-27 b illustrate an alternative embodiment for mounting thechip.

FIG. 28 illustrates an agitation system.

FIG. 29 illustrates an alternative embodiment of the agitation system.

FIG. 30 illustrates another embodiment of the agitation system.

FIG. 31 illustrates an alternative embodiment of a chip packagingdevice.

FIG. 32 illustrates side-views of the chip packaging device of FIG. 31.

FIGS. 33-35 illustrate in greater detail the chip packaging device ofFIG. 31.

FIG. 36 illustrates a further alternative embodiment of a chip packagingdevice.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Contents

I. Definitions

II. General

III. Details of One Embodiment of Invention

-   -   a. Chip Package    -   b. Assembly of Chip Package    -   c. Chip Attachment

IV. Details on Alternative Embodiments

-   -   a. Chip Package    -   b. Chip Attachment    -   c. Fluid Retention    -   d. Chip Orientation    -   e. Parallel Diagnostics

V. Details of an Agitation System

I. Definitions

The following terms are intended to have the following general meaningsas they are used herein:

-   -   1. Probe: A probe is a surface-immobilized molecule that is        recognized by a particular target and is sometimes referred to        as a ligand. Examples of probes that can be investigated by this        invention include, but are not restricted to, agonists and        antagonists for cell membrane receptors, toxins and venoms,        viral epitopes, hormones (e.g., opioid peptides, steroids,        etc.), hormone receptors, peptides, enzymes, enzyme substrates,        cofactors, drugs, lectins, sugars, oligonucleotides or nucleic        acids, oligosaccharides, proteins, and monoclonal antibodies.    -   2. Target: A target is a molecule that has an affinity for a        given probe and is sometimes referred to as a receptor. Targets        may be naturally occurring or manmade molecules. Also, they can        be employed in their unaltered state or as aggregates with other        species. Targets may be attached, covalently or noncovalently,        to a binding member, either directly or via a specific binding        substance. Examples of targets which can be employed by this        invention include, but are not restricted to, antibodies, cell        membrane receptors, monoclonal antibodies and antisera reactive        with specific antigenic determinants (such as on viruses, cells        or other materials), drugs, oligonucleotides or nucleic acids,        peptides, cofactors, lectins, sugars, polysaccharides, cells,        cellular membranes, and organelles. Targets are sometimes        referred to in the art as anti-probes or anti-ligands. As the        term “targets” is used herein, no difference in meaning is        intended A “Probe Target Pair” is formed when two macromolecules        have combined through molecular recognition to form a complex.

II. General

The present invention provides economical and efficient packagingdevices for a substrate having an array of probes fabricated thereon.The probe arrays may be fabricated according to the pioneeringtechniques disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO92/10092, or U.S. application Ser. No. 08/249,188 filed May 24, 1994,already incorporated herein by reference for all purposes. According toone aspect of the techniques described therein, a plurality of probearrays are immobilized at known locations on a large substrate or wafer.

FIG. 1 a illustrates a wafer 100 on which numerous probe arrays 110 arefabricated. The wafer 100 may be composed of a wide range of material,either biological, nonbiological, organic, inorganic, or a combinationof any of these, existing as particles, strands, precipitates, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates, slides, etc. The wafer may have any convenient shape, such as adisc, square, sphere, circle, etc. The wafer is preferably flat but maytake on a variety of alternative surface configurations. For example,the wafer may contain raised or depressed regions on which a sample islocated. The wafer and its surface preferably form a rigid support onwhich the sample can be formed. The wafer and its surface are alsochosen to provide appropriate light-absorbing characteristics. Forinstance, the wafer may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof. Other materials with which thewafer can be composed of will be readily apparent to those skilled inthe art upon review of this disclosure. In a preferred embodiment, thewafer is flat glass or single-crystal silicon.

Surfaces on the solid wafer will usually, though not always, be composedof the same material as the wafer. Thus, the surface may be composed ofany of a wide variety of materials, for example, polymers, plastics,resins, polysaccharides, silica or silica-based materials, carbon,metals, inorganic glasses, membranes, or any of the above-listed wafermaterials.

Wafer 100 includes a plurality of marks 145 that are located in streets150 (area adjacent to the probe arrays). Such marks may be used foraligning the masks during the probe fabrication process. In effect, themarks identify the location at which each array 110 is to be fabricated.The probe arrays may be formed in any geometric shape. In someembodiments, the shape of the array may be squared to minimize wastedwafer area. After the probe arrays have been fabricated, the wafer isseparated into smaller units known as chips. The wafer, for example, maybe about 5×5 inches on which 16 probe arrays, each occupying an area ofabout 12.8 cm², are fabricated.

FIG. 1 b illustrates a chip that has been separated from the wafer. Asillustrated, chip 120 contains a probe array 110 and a plurality ofalignment marks 145. The marks serve multiple functions, such as: 1)aligning the masks for fabricating the probe arrays, 2) aligning thescriber for separating the wafer into chips, and 3) aligning the chip tothe package during the attachment process. In some embodiments, suchchips may be of the type known as Very Large Scale Immobilized PolymerSynthesis (VLSIPS™) chips.

According to a specific embodiment, the chip contains an away of geneticprobes, such as an array of diverse RNA or DNA probes. In someembodiments, the probe array will be designed to detect or study agenetic tendency, characteristic, or disease. For example, the probearray may be designed to detect or identify genetic diseases such ascystic fibrosis or certain cancers (such as P53 gene relevant to somecancers), as disclosed in U.S. patent application Ser. No. 08/143,312,already incorporated by reference.

According to one embodiment, the wafer is separated into a plurality ofchips using a technique known as scribe and break FIG. 2 a illustrates afully programmable computer controlled scribe and break device, which insome embodiments is a DX-III Scriber breaker manufactured by DynatexInternational™. As shown, the device 200 includes a base 205 with arotation stage 220 on which a wafer is mounted. The rotation stageincludes a vacuum chuck for fixing the wafer thereon. A stepper motor,which is controlled by the system, rotates stage 220. Located above thestage is a head unit 230 that includes a camera 232 and cutter 231. Headunit 230 is mounted on a dual-axis frame. The camera generates an imageof the wafer on video display 210. The video display 210 includes across hair alignment mark 215. The camera, which includes a zoom lensand a fiber optic light, allows a user to inspect the wafer on the videodisplay 210. A control panel 240 is located on the base for operatingdevice 200.

In operation, a user places a wafer 100 on a frame 210 as illustrated inFIG. 2 b. The surface of frame 210 is composed of a flexible and stickymaterial. The tackiness of the frame prevents the chips from beingdispersed and damaged during the breaking process. Frame 210 may be apick and place frame or a hoop that is commonly associated withfabrication of semiconductors. Referring back to FIG. 2 a, a user placesthe frame with the wafer on the rotation stage 220. In some embodiments,the frame is held on the rotation stage by vacuum pressure. The userthen aligns the wafer by examining the image displayed on the videodisplay 210.

According to one embodiment, wafer alignment is achieved in two steps.First, using the control panel 240, the user rotates stage 220. Thestage is rotated until streets 150 are aligned with the cross hair 215on the display, as illustrated in FIG. 2 c. Next, the user moves thecutter until it is aligned at the center of one of the streets. Thisstep is performed by aligning horizontal line 216 of the cross hairbetween alignment marks 145, as shown in FIG. 2 d.

Once the cutter is aligned, the user instructs the device to scribe thewafer. In some embodiments, various options are available to the user,such as scribe angle, scribe pressure, and scribe depth. Theseparameters will vary depending on the composition and/or thickness ofthe wafer. Preferably, the parameters are set to scribe and break thewafer without causing any damage thereto or penetrating through theframe. The device repeatedly scribes the wafer until all the streets inone axis have been scribed, which in one embodiment is repeated 5 times(a 4×4 matrix of probe arrays). The user then rotates the stage 90° toscribe the perpendicular streets.

Once the wafer has been scribed, the user instructs the device to breakor separate the wafer into chips. Referring back to FIG. 2 a, the device200 breaks the wafer by striking it beneath the scribe with an impulsebar located under the rotation table 220. The shock from the impulse barfractures the wafer along the scribe. Since most of the force isdissipated along the scribe, device 200 is able to produce high breakingforces without exerting significant forces on the wafer. Thus, the chipsare separated without causing any damage to the wafer. Once separated,the chips are then packaged. Of course, other more conventionaltechniques, such as the sawing technique disclosed in U.S. Pat. No.4,016,855, incorporated herein by reference for all purposes, may beemployed.

III. Details of One Embodiment of the Invention

a. Chip Package

FIG. 3 illustrates a device for packaging the chips. Package 300contains a cavity 310 on which a chip is mounted. The package includesinlets 350 and 360 which communicate with cavity 310. Fluids arecirculated through the cavity via inlets 350 and 360. A septum, plug, orother seal may be employed to seal the fluids in the cavity. Alignmentholes 330 and 335 may be provided for alignment purposes. In someembodiments, the package may include a non-flush edge 320. In somedetection systems, the packages may be inserted into a holder similar toan audio cassette tape. The asymmetrical design of the package willassure correct package orientation when inserted into the holder.

FIG. 4 illustrates one embodiment of the package. As shown in FIG. 4,the chip package is manufactured by mating two substantiallycomplementary casings 410 and 420 to form finished assembly 300.Preferably, casings 410 and 420 are made from injection molded plastic.Injection molding enables the casings to be formed inexpensively. Also,assembling the package from two parts simplifies the construction ofvarious features, such as the internal channels for introducing fluidsinto the cavity. As a result, the packages may be manufactured at arelatively low cost.

FIGS. 5 a-5 b show the top casing 410 in greater detail. FIG. 5 a showsa top view and FIG. 5 b shows a bottom view. Referring to FIG. 5 a, topcasing 410 includes an external planar surface 501 having a cavity 310therein. In some embodiments, the surface area of casing 410sufficiently accommodates the cavity. Preferably, the top casing is ofsufficient size to accommodate identification labels or bar codes inaddition to the cavity. In a specific embodiment, the top casing isabout 1.5″ wide, 2″ long, and 0.2″ high.

Cavity 310 is usually, though not always, located substantially at thecenter of surface 501. The cavity may have any conceivable size, shape,or orientation. Preferably, the cavity is slightly smaller than thesurface area of the chip to be placed thereon and has a volumesufficient to perform hybridization. In one embodiment, the cavity maybe about 0.58″ wide, 0.58″ long, and 0.2″ deep.

Cavity 310 may include inlets 350 and 360. Selected fluids areintroduced into and out of the cavity via the inlets. In someembodiments, the inlets are located at opposite ends of the cavity. Thisconfiguration improves fluid circulation and regulation of bubbleformation in the cavity. The bubbles agitate the fluid, increasing thehybridization rate between the targets and complementary probesequences. In one embodiment, the inlets are located at the top andbottom end of the cavity when the package is oriented vertically such asat the opposite corners of the cavity. Locating the inlet at the highestand lowest positions in the cavity facilitates the removal of bubblesfrom the cavity.

FIG. 5 c illustrates an alternative embodiment in which cavity 310 isoriented such that the edges of the cavity 310 and the casing 410 arenon-parallel. This configuration allows inlets 350 and 360 to besituated at the absolute highest and lowest locations in the cavity whenthe package is vertically oriented. As a result, bubbles or fluiddroplets are prevented from being potentially trapped in the cavity.

Referring back to FIG. 5 a, a depression 550 surrounds the cavity. Insome embodiments, a ridge 560 may be provided at the edge of thedepression so as to form a trough. The ridge serves to support the chipabove the cavity. To attach the chip to the package, an adhesive may bedeposited in the trough. This configuration promotes efficient use ofchip surface area, thus increasing the number of chips yielded from awafer.

Top casing 410 includes alignment holes 330 and 335. In someembodiments, holes 330 and 335 are different in size to ensure correctorientation of the package when mounted on an alignment table.Alternatively, the holes may have different shapes to achieve thisobjective. Optionally, the holes taper radially inward from surface 501toward 502 to reduce the friction against alignment pins while stillmaintaining adequate contact to prevent slippage.

Referring to FIG. 5 b, channels 551 and 561 are optionally formed oninternal surface 502. Channels 551 and 561 communicate with inlets 350and 360 respectively. A depression 590 is formed below cavity. Accordingto some embodiments, the shape of depression 590 is symmetrical to thecavity with exception to corners 595 and 596, which accommodate theinlets. The depth of depression 590 may be, for example, about 0.7″. Asa result, the bottom wall of the cavity is about 0.05″ thick. Depression590 may receive a temperature controller to monitor and maintain thecavity at the desired temperature. By separating the temperaturecontroller and cavity with a minimum amount of material, the temperaturewithin the cavity may be controlled more efficiently and accurately.Alternatively, channels may be formed on surface 502 for circulating airor water to control the temperature within the cavity.

In some embodiments, certain portions 595 of internal surface 502 may beeliminated or cored without interfering with the structural integrity ofthe package when assembled. Coring the casing reduces the wallthickness, causing less heat to be retained during the injection moldingprocess; potential shrinkage or warpage of the casing is significantlyreduced. Also, coring decreases the time required to cool the casingduring the manufacturing process. Thus, manufacturing efficiency isimproved.

In one embodiment, the top casing and bottom casing are mated togetherusing a technique known as acoustic or ultrasonic welding. Accordingly,“energy directors” 510 are provided. Energy directors are raised ridgesor points, preferably v-shaped, that are used in an acoustic weldingprocess. The energy directors are strategically located, for example, toseal the channels without interfering with other features of the packageand to provide an adequate bond between the two casings. Alternatively,the casings may be mated together by screws, glue, clips, or othermating techniques.

FIG. 6 shows a cross sectional view of the cavity 310 with chip 120mounted thereon in detail. As shown, a depression 550 is formed aroundcavity 310. The depression includes a ridge 560 which supports chip 120.The ridge and the depression create a trough around cavity 310. In someembodiments, the trough is sufficiently large to receive an adhesive 630for attaching the chip to the package. In one embodiment, the trough isabout 0.08″ wide and 0.06″ deep. When mounted, the edge of the chipprotrudes slightly beyond ridge 550, but without contacting side 625 ofthe depression. This configuration permits the adhesive to be dispensedonto the trough and provides adequate surface area for the adhesive toattach chip 120 to the package.

According to some embodiments, the back surface 130 of chip 120 is atleast flush or below the plane formed by surface 501 of casing 410. As aresult, chip 120 is shielded by surface 501 from potential damage. Thisconfiguration also allows the packages to be easily stored with minimalstorage area since the surfaces are substantially flat.

Optionally, the bottom of the cavity includes a light absorptivematerial, such as a glass filter or carbon dye, to prevent impinginglight from being scattered or reflected during imaging by detectionsystems. This feature improves the signal-to-noise ratio of such systemsby significantly reducing the potential imaging of undesired reflectedlight.

FIG. 7 shows the internal surface of bottom casing 420 in greaterdetail. As shown, the bottom casing 420 is substantially planar andcontains an opening 760 therein. Preferably, the casing 420 is slightlywider or slightly longer than the top casing. In one embodiment, casing420 is about 1.6″ wide, 2.0″ long, and 0.1″ deep, which creates anon-flush edge on the finish assembly. As previously mentioned, thisdesign ensures that the package is correctly oriented when mounted ontothe detection systems.

In some embodiments, opening 760 is spatially located at about thedepression below the cavity. The opening also has substantially the samegeometric configuration as the depression to allow the temperaturecontroller to contact as much of the bottom of the cavity as possible.

Internal surface 701 of casing 420 includes depressions 730 and 740. Aport 731 is located in depression 730 and a port 741 is located indepression 740. Ports 731 and 741 communicate with channels on the topcasing (350 and 360 in FIG. 5 b) when the package is assembled. A seal790, which may be a septum composed of rubber, teflon/rubber laminate,or other sealing material is provided for each depression. The septummay be of the type commonly used to seal and reseal vessels when aneedle is inserted into the septum for addition/removal of fluids. Theseptums, when seated in the depressions, extend slightly above surface,which in some embodiments is about 0.01″.

This design causes casings 410 and 420 to exert pressure on the septum,forming a seal between the ports and the channels. The seal ismaintained even after fluid is injected into the cavity since thepressure immediately forces the septum to reseal itself after the needleor other fluid injecting means is removed from the port. Thus, anefficient and economical seal for retaining fluid in the cavity isprovided.

Also, casing 420 includes the complementary half alignment holes 330 and335, each tapering radially inward from the external surface. Further,certain areas 765 on internal surface 701 may be cored, as similar tothe internal surface of the top casing.

FIG. 31 is a simplified illustration of an alternative embodiment of achip packaging device 3100 according to the present invention. The chippackaging device includes a plurality of casings 3200, 3300, and 3400.The casings may be defined as a top casing 3200, a middle casing 3300,and a bottom casing 3400. The casings are made of known plasticmaterials such as ABS plastic, polyvinylchloride, polyethylene, productssold under the trademarks TEFLON™ and KALREZ™ and the like, amongothers. Preferably, the casings can be made by way of injection moldingand the like. Assembling the chip packaging device from three casingssimplifies construction for the fabrication of internal channels and thelike, and can also be made at a relatively low cost.

Support structures (or alignment holes) exist at selected locations ofthe chip packing device. The support structures can be used to mount orposition the chip packaging device to an apparatus, e.g., scanner or thelike. In an embodiment, the top casing 3200 includes support structures3201 and 3203 on each side of a center opening 3209. The middle casing3300 includes similar support structures 3313 and 3315 which arecomplementary to the support structures 3201 and 3203, respectively, inthe top casing. The bottom casing also includes similar supportstructures 3403 and 3401, respectively, which are complementary to thesupport structures in the top casing and the middle casing. As shown,each of the support structures on each side of the center opening alignwith each other. Each support structure is, for example, an aperturethrough the casing. The aperture includes an outer periphery defined bya geometrical shape which may be round, rectangular, trapezoidal,hexagonal, or the like.

The present chip packaging device assembles with use of complementaryalignment pins and bores on the casings. By way of alignment pins (notshown), the top casing aligns with and inserts into alignment bores3301, 3303 in the middle casing 3300. Alternatively, the middle casingcan have alignment pins or the like and the top casing has the alignmentbores or the like. The bottom casing includes alignment pins 3407 and3409 which align to and insert into alignment bores (not shown) inbottom portions of the middle casing. The use of alignment bores andpins provide for ease in assembly of the chip carrier. Upon assembly,the alignment bores and pins on the casings prevent the casings frommoving laterally relative to each other.

A center opening 3209 in the top casing overlies a center portion 3317of the middle casing 3300. The center portion 3317 of the middle casingincludes an inner annular region (or cavity edges) with a bottom portionwhich is preferably a flat bottom portion. The flat bottom portion ofthe middle casing and portions of the bottom casing including edgesdefine a cavity 3405. A chip is placed overlying an underlying portionof the cavity 3407.

Optionally, a temperature control mechanism such as a heater, a cooler,or a combination thereof is disposed into the center opening against thebottom portion of the middle casing. The temperature control mechanismcan be any suitable thermally controlled element such as a resistiveelement, a temperature controlled block or mass, thermoelectric modules,or the like. The temperature control mechanism transfers heat viaconduction to the bottom center portion, which transfers heat to, forexample, fluid in the cavity or the chip. Alternatively, the temperaturecontrol mechanism sinks heat away from, for example, fluid in the cavityor the chip through the bottom center portion. The temperature controlmechanism maintains a selected temperature in the cavity. Thetemperature control mechanism also includes a temperature detectiondevice such as a thermocouple which provides signals corresponding totemperature readings. A controller receives the signals corresponding tothe temperature readings, and adjusts power output to the temperaturecontrol mechanism to maintain the selected temperature.

The top casing 3200 also includes channels 3205 and 3207 for fluidtransfer. The channels 3205 and 3207 communicate with annular regions3309 and 3311, respectively, on the middle casing 3300 for fluidtransfer. A septum, a plug, an o-ring, a gasket, or the like via annularregions 3309 and 3311 seals fluids within the top casing channels 3205and 3207 and the middle casing. The bottom casing includes channels 3411and 3413 in communication with channels 3307 and 3305, respectively. Aseptum, a plug, an o-ring, a gasket, or the like seals the fluids withinthe bottom casing channels 3411 and 3413 and the middle casing channels3305 and 3307.

The chip packaging device provides an even distribution of fluid (orfluid flow) through the cavity over a top surface (or inner or activesurface) of the chip. For example, a selected fluid enters channel 3207,flows through channel 3307, changes direction and flows through channel3411, and evenly distributes into the cavity 3405 over the top surfaceof the chip. As previously noted, the cavity is defined by the flatbottom portion and cavity edges. A selected fluid exits the cavity byway of channel 3413, channel 3305, and channel 3205. The fluid flow overthe top surface of the chip is preferably laminar, but may also beturbulent, a combination thereof or the like. By way of the present chippackaging device, a substantial portion of turbulent flow remains at anupper portion of the channel 3411, and does not enter the cavity.

Preferably, a selected fluid enters the cavity by way of channel 3205,channel 3305, and channel 3413. The selected fluid exits the cavitythrough channel 3411, channel 3307, and channel 3207. In a preferredembodiment, the fluid flows against the direction of gravity through thecavity. Of course, other fluid flow routes may also be employeddepending upon the particular application.

FIG. 32 illustrates an assembled chip packaging device 3100 according tothe present invention. As shown are a top-view 3200, a side-view 3500, abottom-view 3400, and a front-view 3600 of the assembled chip packagingdevice 3100. The assembled chip packaging device 3100 includes thebottom casing 3400, the middle casing 3300, and the top casing 3200.

The top-view 3200 of the top casing includes alignment structures 3205,3215 surrounding opening 3209. The opening 3209 includes a bevelledannular region 3211 surrounding the periphery of the channel 3209. Thealignment bores 3203 and 3201 also include bevelled annular regions 3213and 3215, respectively. A bevelled annular region 3217, 3221 alsosurrounds each fluid channel 3205, 3207 to assist with fluid flowtherethrough.

The bottom-view 3400 of the bottom casing includes alignment structures3401, 3403 surrounding the cavity 3405. The cavity includes a flatbottom peripheral portion 3415, a bevelled portion 3417 extending fromthe flat bottom peripheral portion, and a flat upper portion 3419surrounding the bevelled portion. The chip includes an outer peripherywhich rests against the flat bottom peripheral portion 3415. Thebevelled portion aligns the chip onto the flat bottom peripheral portion3415. Similar to the previous embodiments, the top casing extendsoutside 3421 the middle and bottom casings.

The cavity 3405 is preferably located at a center of the bottom casing,but may also be at other locations. The cavity may be round, square,rectangular, or any other shape, and orientation. The cavity ispreferably smaller than the surface area of the chip to be placedthereon, and has a volume sufficient to perform hybridization and thelike. In one embodiment, the cavity includes dimensions such as a lengthof about 0.6 inch, a width of about 0.6 inch and a depth of about 0.07inch.

In a preferred embodiment, the bottom casing with selected cavitydimensions may be removed from the middle and top casings, and replacedwith another bottom casing with different cavity dimensions. This allowsa user to attach a chip having a different size or shape by changing thebottom casing, thereby providing ease in using different chip sizes,shapes, and the like. Of course, the size, shape, and orientation of thecavity will depend upon the particular application.

FIGS. 33-35 illustrate in greater detail the chip packaging device ofFIG. 31. FIG. 33 illustrates simplified top-view 3260 and bottom-view3250 diagrams of the top casing 3200. As shown, the reference numeralsrefer to the same elements as the top casing of FIG. 31. FIG. 34illustrates a simplified top-view 3350 and bottom-view 3360 diagrams ofthe middle casing 3300. As shown, the reference numerals refer to thesame elements as the middle casing of FIG. 31. In addition, thebottom-view of the casing includes a substantially smooth and planarbottom surface 3361. A portion of the bottom surface defines an upperportion of the cavity. But the bottom surface can also be textured,ridged, or the like to create turbulence or a selected fluid flowthrough the cavity. The bottom surface is preferably a hydrophobicsurface which enhances laminar flow through the cavity. Of course, thetype of bottom surface depends upon the particular application.

FIG. 35 illustrates simplified top-view 3460 and bottom-view 3450diagrams of the bottom casing 3400. As shown, the reference numeralsrefer to the same elements as the bottom casing of FIG. 31. In anembodiment, fluid from channel 3305 changes direction at an upperportion 3431 of the channel and flows to a lower portion 3433 of thechannel. Fluid evenly distributes from the lower portion 3433 via afluid distribution point 3435. The distributed fluid evenly passes overa slanted edge (or bevelled edge) 3437 which drops fluid evenly to a topsurface of the chip in the cavity. By way of slanted edge 3427 whichslopes up to a fluid concentration point 3425, fluid leaves the cavityand enters the channel 3411. In particular, fluid leaves the cavity andenters a lower portion 3423 of the channel, flows through the channel,and changes directions at an upper portion 3421 of the channel. Eachchannel includes a length L and a width W. The distribution point andthe concentration point are positioned at a distance away from thecavity to substantially prevent turbulence from forming in the cavity,and in particular over the top surface of the chip. The channels areeach angled at an angle Θ ranging from about 2 degrees to about 90degrees, but is preferably about 5 degrees to about 45 degrees. Theangle enhances an even distribution of laminar flow into the cavity. Ofcourse, the exact angle, channel shape, and dimensions depend upon theparticular application.

FIG. 36 illustrates a simplified cross-sectional view of an alternativeembodiment 3600 of the chip packaging device. The chip packaging deviceincludes the three casings 3200, 3300, and 3400 of the previousembodiment, and also includes hollow pins, needles, or the like 3601 and3603. Each of the pins transfers a selected fluid to and from the cavity3405. Preferably, each pin 3601 includes an external opening 3609, atubular region 3611, an inner opening 3607, a pointed tip 3605, andother elements. The pin is made from a suitable material such as aglass, a stainless steel or any other high quality material to transferfluids to and from the cavity 3405.

In a preferred embodiment, each pin is inserted into its channel region3205 or 3207. A point on the pin tip pierces through, for example, aseptum at an annular region 3309 or 3311. A selected fluid travelsthrough pin 3603 (through channel 3205 and at least a portion of 3305),enters the upper region of channel 3413, and into the cavity 3405. Theselected fluid travels from the cavity, through pin 3601, and to theexternal apparatus. Alternatively, the selected fluid enters the cavityvia pin 3601 and exits the cavity via pin 3603. The selected fluid mayalso enter the cavity via pin and exit the cavity through the channelswithout use of a pin. The selected fluid may further enter the cavitythrough the channels without use of a pin and exit through a pin. Ofcourse, the particular pin used and fluid flow will depend upon theapplication.

It should be noted that the even distribution of fluid flow through thecavity prevents “hot spots” from occurring in the cavity. For example,the even distribution of fluid through the cavity by way of the previousembodiment substantially prevents fluid from becoming substantiallyturbulent at certain locations. This prevents “hot spots” caused by suchturbulent fluid. The hot spots are often caused by higher chemicalactivity or exothermic reactions and the like by way of turbulence insuch certain locations.

b. Assembly of Chip Package

According to one embodiment, the top and bottom casing are attached by atechnique known as ultrasonic or acoustic welding. FIG. 8 a is aschematic diagram of acoustic welding system used for assembling thepackage. In some embodiments, the welding system 800 is a HS Dialogultrasonic welder manufactured by Herrmann Ultrasonics Inc. System 800includes a platform 850 mounted on base 810. Platform 850 accommodatesthe top and bottom casings during the assembling process.

An acoustic horn 860 is mounted on a frame above platform 850. The horntranslates vertically (toward and away from platform 850) on the frameby air pressure. The horn is connected to a frequency generator 870,which in some embodiments is a 20 KHz generator manufactured by HerrmannUltrasonics Inc. System 800 is controlled by a controller 880, which,for example, may be a Dialog 2012 manufactured by Herrmann UltrasonicsInc. Controller 880 may be configured to accept commands from a digitalcomputer system 890. Computer 890 may be any appropriately programmeddigital computer of the type that is well known to those skilled in theart such as a Gateway 486DX operating at 33 MHz.

FIG. 8 b illustrates platform 850 in greater detail. The platform 850 issubstantially planar and includes alignment pins 851 and 852. Alignmentpins 851 and 852 are used to align both the top and bottom casingsduring the welding process. In some embodiments, a pad 890, which may becomposed of silicone rubber or other energy absorbing material, islocated on platform 850 to prevent damage to the package duringassembly.

FIG. 9 a illustrates the acoustic welding system in operation. As shown,bottom casing 420, having a septum 790 seated in each depression, ismounted onto platform table 850 and held in place by alignment pins. Topcasing 410 is then aligned above the bottom casing with alignment pins.The system then commences the welding process by lowering horn 860 untilit contacts the top surface of casing 410.

FIG. 9 b illustrates the casing and horn in detail. As shown, the horn860 presses against top casing 410, thereby forcing energy directors 510to interface with bottom casing 420. The system then activates thefrequency generator, causing the welding horn to vibrate.

FIG. 9 c illustrates in detail the energy directors during the weldingprocess. As shown in step 9001, welding horn 860 forces energy directors510 against bottom casing 420. At step 9002, the system vibrates thewelding horn, which in some embodiments is at 20 KHz. The energygenerated by the horn melts the energy directors. Simultaneously, thehorn translates downward against the package. At step 9003, the pressureexerted by the horn causes the energy directors to fuse with the bottomcasing. At step 9004, the welding process is completed when the hornreaches its weld depth, for example, of about 0.01″. Of course, thevarious welding parameters may be varied, according to the compositionof the materials used, to achieve optimum results.

c. Chip Attachment

According to some embodiments, an ultraviolet cured adhesive attachesthe chip to the package. FIG. 10 schematically illustrates an adhesivedispensing system used in attaching the chip. The dispensing system 1000includes an attachment table 1040 to accommodate the package during theattachment process. A chip alignment table 1050 for aligning the chip islocated adjacent to attachment table 1040. A head unit 1030 fordispensing the adhesive is located above tables 1040 and 1050. The headunit 1030 also includes a camera that generates an output to videodisplay 1070. Video display 1070, in some embodiments, includes a crosshair alignment mark 1071. The head unit is mounted on a dual-axis (x-y)frame for positioning during alignment and attachment of the chip. Theoperation of the dispensing system is controlled by a computer 1060,which in some embodiments may be Gateway 486DX operating at 33 MHz.

FIG. 11 illustrates the attachment table in greater detail. Theattachment table 1040 has a substantially flat platform 1110 supportedby a plurality of legs 1105. Alignment pins 1115 and 1116, which securethe package during the attachment process, are located on the surface ofplatform 1110.

Optionally, a needle 1120 is provided. Needle 1120 includes a channel1121 and is connected to a vacuum pump. In operation, the needle isinserted into one of the ports of the package in order to generate avacuum in the cavity. The vacuum pressure secures the chip to thepackage during the attachment process.

FIG. 12 a shows table 1050 in greater detail. Table 1050 includes asubstantially flat platform 1210 having a depression 1240 for holding achip. In some embodiments, a port 1241 is provided in depression 1240.Port 1241 is connected to a vacuum pump which creates a vacuum in thedepression for immobilizing the chip therein. Platform 1210 is mountedon a combination linear rotary stage 1246, which in some embodiments maybe a model 26LR manufactured by DARDAL, and a single axis translationstage 1245, which may be a model CR2226HSE2 manufactured by DARDAL.

FIG. 12 b illustrates depression 1240 in greater detail. As shown, aledge 1241 surrounds the depression 1240. Ledge 1241 supports the chipwhen it is placed above depression 1240. Since the chips are placed overthe depression with the probes facing the table, this design protectsthe probes from being potentially damaged during alignment.

FIG. 13 illustrates the head unit 1030 in greater detail. As shown, thebead unit 1030 includes a camera assembly 1320 that generates an outputto a video display. A light 1360 is provided to enable the camera tofocus and image an object of interest. The bead unit also includes anultraviolet light 1350 for curing the adhesive, a vacuum pickup 1330 formoving chip during the attachment process, and an adhesive dispenser1340.

In operation, a chip package is placed onto table 1040. As previouslydescribed, the alignment pins on the table immobilize the package. Theuser begins the chip attachment process by calibrating the head unit.This may be done by moving the camera above the package and aligning itwith a mark on the package, as shown in FIG. 14 a. For convenience, oneof the alignment pins may be used as an alignment mark. FIG. 14 billustrates a typical image 1440 generated by the camera during thisstep. As shown, the head unit is not aligned with pin 1480. To align thehead unit, the user translates it in both the x and y direction untilpin 1480 is located at the intersection 1477 of the cross hair on thevideo display, as illustrated in FIG. 14 c.

Next, the chip is inserted into the depression on the chip alignmenttable. FIG. 14 c is a flow chart indicating the steps for aligning thechip. At step 1410, the system positions the camera (head unit) aboveone of the chip's alignment marks. The camera images the alignment markon the video display. At this point, the mark is normally misaligned(i.e., the mark is not located at the intersection of the cross hairalignment mark). At step 1420, the user adjusts the chip alignment tablein both the x and y direction until the mark is substantially located atthe intersection of the cross hair. Since no rotational adjustments weremade, the mark may be misaligned angularly.

At step 1430, the user instructs the system to move the camera above asecond alignment mark, which usually is at an opposite corner of thechip. Again, an image of the alignment mark is displayed. At this stage,the alignment mark is probably misaligned in the x, y, and angulardirections. At step 1440, the user adjusts the rotational stage,x-stage, and y-stage, if necessary, to align the mark with the crosshair on the video display. In instances where the rotational stage hasbeen rotated, the first alignment mark will become slightly misaligned.To compensate for this shift, the user repeats the alignment processbeginning at step 1450 until both marks are aligned. Of course, imageprocessing techniques may be applied for automated head unit and chipalignment.

FIG. 15 a is an example of an image displayed by the video screen duringstep 1410. As shown, the first alignment mark (lower left corner of thechip) is not aligned with the cross hair marking. FIG. 15 b exemplifiesan image of the first alignment mark after adjustments were made by theuser. FIG. 15 c illustrates a typical image displayed by video screenduring step 1430. As illustrated, the second alignment mark (upper rightcorner of the chip) is misaligned in the x, y, and angular directions.FIG. 15 d illustrates an image of the second mark following initialadjustments by the user at step 1440. FIG. 15 e illustrates theorientation of the second alignment mark after the chip has beenaligned.

Once the chip is aligned, the vacuum holding the chip on the attachmenttable is released. Thereafter, the pickup on the head unit removes thechip from the table and aligns it on the cavity of the package. In someembodiments, the chip is mated to the pickup by a vacuum.

Optionally, the user may check to ensure that the chip is correctlyaligned on the cavity by examining the chip's alignment marks with thecamera. If the chip is out of position, the chip is removed andrealigned on the alignment table. If the chip is correctly positioned,the system deposits an adhesive by moving the dispenser along the troughsurrounding the cavity. In some embodiments, the vacuum is releasedbefore depositing the adhesive in the trough. This step is merelyprecautionary and implemented to ensure that the vacuum does not causeany adhesive to seep into the cavity. Once the adhesive is deposited,the system reexamines the chip to determine if the adhesive had movedthe chip out of position. If the chip is still aligned, the head unitlocates the ultraviolet light above the adhesive and cures it for a timesufficient to harden the adhesive, which in one embodiment is about 10seconds. Otherwise, the chip is realigned.

Upon completion, the chip package will have a variety of uses. Forexample, the chip package will be useful in sequencing genetic materialby hybridization. In sequencing by hybridization, the chip package ismounted on a hybridization station where it is connected to a fluiddelivery system. Such system is connected to the package by insertingneedles into the ports and puncturing the septums therein. In thismanner, various fluids are introduced into the cavity for contacting theprobes during the hybridization process.

Usually, hybridization is performed by first exposing the sample with aprehybridization solution. Next, the sample is incubated under bindingconditions with a solution containing targets for a suitable bindingperiod. Binding conditions will vary depending on the application andare selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al., Molecular Cloning: ALaboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Bergerand Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;Young and Davis (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80: 1194, whichare incorporated herein by reference. In some embodiments, the solutionmay contain about 1 molar of salt and about 1 to 50 nanomolar oftargets. Optionally, the fluid delivery system includes an agitator toimprove mixing in the cavity, which shortens the incubation period.Finally, the sample is washed with a buffer, which may be 6×SSPE buffer,to remove the unbound targets. In some embodiments, the cavity is filledwith the buffer after washing the sample.

Thereafter, the package may be aligned on a detection or imaging system,such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.) orU.S. patent application Ser. No. 08/495,889 (Attorney Docket Number11509-117), already incorporated herein by reference for all purposes.Such detection systems may take advantage of the package's asymmetry(i.e., non-flush edge) by employing a holder to match the shape of thepackage specifically. Thus, the package is assured of being properlyoriented and aligned for scanning. The imaging systems are capable ofqualitatively analyzing the reaction between the probes and targets.Based on this analysis, sequence information of the targets isextracted.

IV. Details on Alternative Embodiments

a. Chip Package Orientation

FIGS. 16 a-16 b illustrate an alternative embodiment of the package.FIG. 16 a shows a top view and FIG. 16 b shows a bottom view. As shownin FIG. 16 a, a cavity 1620 is located on a top surface 1610 of thepackage body 1600. The body includes alignment holes 1621 and 1622 thatare used, for example, in mating the chip to the package. Optionally, aplurality of ridges 1690 is located at end 1660 of the body. Thefriction created by ridges 1690 allows the package to be handled easilywithout slippage.

The body also includes two substantially parallel edges 1630 and 1640.As shown, edge 1640 is narrowed at end 1665 to create an uneven edge1645. The asymmetrical design of the body facilitates correctorientation when mounted onto detection systems. For example, detectionsystems may contain a holder, similar to that of an audio cassette tape,in which end 1665 is inserted.

Referring to FIG. 16 b, ports 1670 and 1671 communicate with cavity1620. A seal is provided for each port to retain fluids in the cavity.Similar to the top surface, the bottom surface may optionally include aplurality of ridges 1690 at end 1660.

FIGS. 17 a-17 b illustrate an alternative embodiment of the package.FIG. 17 a shows a top view and FIG. 17 b shows a bottom view. Referringto FIG. 17 a, a cavity 1720 is located on a top surface 1710 of thepackage body 1700. The body may be formed in the shape of a disk withtwo substantially parallel edges 1730 and 1740. Alignment holes 1721 and1722, which may be different in size or shape, are located on the body.In some embodiments, the package is inserted like an audio cassette tapeinto detection systems in a direction parallel to edges 1730 and 1740.Edges 1730 and 1740 and alignment holes prevent the package from beinginserted incorrectly into the detection systems.

As shown in FIG. 17 b, ports 1730 and 1740 are located on the bottomsurface 1715 of the package. Ports 1730 and 1740 communicate with cavity1720 and each include a seal 1780 for sealing fluids in the cavity.

b. Chip Attachment

FIG. 18 illustrates an alternative embodiment for attaching the chip tothe package. As shown, two concentric ledges 1810 and 1820 surround theperimeter of cavity 310. Ledge 1820 supports the chip 120 when mountedabove cavity 310. Ledge 1810, which extends beyond chip 120, receives anadhesive 1860 such as ultraviolet cured silicone, cement, or otheradhesive for attaching the chip thereto.

FIG. 19 illustrates another embodiment for attaching the chip to thepackage. According to this embodiment, a ledge 1910 is formed aroundcavity 310. Preferably, the ledge is sufficiently large to accommodatean adhesive 1920 such as an adhesive film, adhesive layer, tape, or anyother adhesive layer. Chip 120 attaches to the package when it contactsthe adhesive film.

FIG. 20 a illustrates yet another embodiment for attaching a chip to thepackage. As shown, a clamp 2010, such as a frame having a plurality offingers 2015, attaches the chip to the package. FIG. 20 b illustrates across sectional view. A ridge 2020 on surface 501 surrounds cavity 310.The ridge includes a ledge 2025 upon which chip 120 rests. Optionally, agasket or a seal 2070 is located between the ledge and chip to ensure atight seal around cavity 310. Clamp 2010 is attached to side 2040 ofridge 2020 and surface 501. In some embodiments, clamp 2010 isacoustically welded to the body. Accordingly, clamp 2010 includes energydirectors 2050 located at its bottom. Alternatively, screws, clips,adhesives, or other attachment techniques may be used to mate clamp 2010to the package. When mated, fingers 2015 secure chip 120 to the package.

FIG. 21 illustrates an alternative embodiment for attaching the chip tothe package. A ridge 2110, having a notch 2115 at or near the top ofridge 2110, encompasses the cavity 310. Chip 120 is wedged and held intoposition by notch 2115. Thereafter, a process known as heat staking isused to mount the chip. Heat staking includes applying heat and force atside 2111 of ridge, thus forcing ridge tightly against or around chip120.

FIG. 22 shows another embodiment of attaching a chip onto a package. Asshown, a channel 2250 surrounds cavity 310. A notch 2240 for receivingthe chip 120 is formed along or near the top of the cavity 310. In someembodiments, a gasket or seal 2270 is placed at the bottom of the notchto ensure a tight seal when the chip is attached. Once the chip islocated at the notch, a V-shaped wedge 2260 its inserted into channel2250. The wedge forces the body to press against chip's edges and seal2260, thus mating the chip to the package. This process is known ascompression sealing.

Other techniques such as insert molding, wave soldering, surfacediffusion, laser welding, shrink wrap, o-ring seal, surface etching, orheat staking from the top may also be employed.

c. Fluid Retention

FIG. 23 shows an alternative embodiment of package that employs checkvalves to seal the inlets. As shown, depressions 2305 and 2315communicate with cavity 310 through inlets 350 and 360. Check valves2310 and 2320, which in some embodiments may be duck-billed checkvalves, are seated in depressions 2305 and 2315. To introduce a fluidinto the cavity, a needle is inserted into the check valve. When theneedle is removed, the check valve reseals itself to prevent leakage ofthe fluid.

FIG. 24 illustrates another package that uses reusable tape for sealingthe cavity 310. As shown, a tape 2400 is located above inlets 350 and360. Preferably, end 2430 of tape is permanently fixed to surface 2480while end 2410 remains unattached. The mid section 2420 of the tape iscomprised of non-permanent adhesive. This design allows inlets to beconveniently sealed or unsealed without completely separating the tapefrom the package.

FIG. 25 illustrates yet another embodiment of the package that usesplugs to retain fluids within the cavity. As shown, depressions 2520 and2530 communicate with cavity 310 via inlets 350 and 360. A plug 2510,which in some embodiment may be composed of rubber or other sealingmaterial, is mated to each of the depressions. Plugs 2510 are easilyinserted or removed for sealing and unsealing the cavity during thehybridization process.

FIG. 26 a illustrates a package utilizing sliding seals for retainingfluids within the cavity. The seals are positioned in slots 2610 thatare located above the inlets. The slots act as runners for guiding theseals to and from the inlets. FIG. 26 b illustrates the seal in greaterdetail. Seal 2640, which may be composed of rubber, teflon rubber, orother sealing material, is mated to each slot 2610. The seal includes ahandle 2650 which extends through the slot. Optionally, the bottom ofthe seal includes an annular protrusion 2645 to ensure mating with inlet350. The inlet is sealed or unsealed by positioning the sealappropriately along the slot. Alternatively, spring loaded balls, rotaryball valves, plug valves, or other fluid retention techniques may beemployed.

d. Chip Orientation

FIGS. 27 a-27 b illustrate an alternative embodiment of the package.FIG. 27 a illustrates a top view and FIG. 27 b shows a cross sectionalview. As shown, package 2700 includes a cavity 2710 on a surface 2705. Achip 2790 having an army of probes 2795 on surface 2791 is mated to thebottom of cavity 2710 with an adhesive 2741. The adhesive, for example,may be silicone, adhesive tape, or other adhesive. Alternatively, clipsor other mounting techniques may be employed. Optionally, the bottom ofthe cavity may include a depression in which a chip is seated.

This configuration provides several advantages such as: 1) permittingthe use of any type of substrate (i.e., non-transparent ornon-translucent), 2) yielding more chips per wafer since the chip doesnot require an edge for mounting, and 3) allowing chips of various sizesor multiple chips to be mated to the package.

A cover 2770 is mated to the package for sealing the cavity. Preferably,cover 2770 is composed of a transparent or translucent material such asglass, acrylic, or other material that is penetrable by light. Cover2270 may be mated to surface 2705 with an adhesive 2772, which in someembodiments may be silicone, adhesive film, or other adhesive.Optionally, a depression may be formed around the cavity such thatsurface 2271 of the cover is at least flush with surface 2705.Alternatively, the cover may be mated to surface 2705 according to anyof the chip attachment techniques described herein.

Inlets 2750 and 2751 are provided and communicate with cavity 2710.Selected fluids are circulated through the cavity via inlets 2750 and2751. To seal the fluids in the cavity, a septum, plug, or other sealmay be employed. In alternative embodiments, any of the fluid retentiontechniques described herein may be utilized.

e. Parallel Hybridization and Diagnostics

In an alternative embodiment, the body is configured with a plurality ofcavities. The cavities, for example, may be in a 96-well micro-titreformat. In some embodiments, a chip is mounted individually to eachcavity according to the methods described above. Alternatively, theprobe arrays may be formed on the wafer in a format matching that of thecavities. Accordingly, separating the wafer is not necessary beforeattaching the probe arrays to the package. This format providessignificant increased throughput by enabling parallel testing of aplurality of samples.

V. Details of an Agitation System

FIG. 28 illustrates an agitation system in detail. As shown, theagitation system 2800 includes two liquid containers 2810 and 2820,which in the some embodiments are about 10 milliliters each. Container2810 communicates with port 350 via tube 2850 and container 2820communicates with port 360 via tube 2860. An inlet port 2812 and a ventport 2811 are located at or near the top of container 2810. Container2820 also includes an inlet port 2822 and a vent 2821 at or near itstop. Port 2812 of container 2810 and port 2822 of container 2820 areboth connected to a valve assembly 2828 via valves 2840 and 2841. Anagitator 2801, which may be a nitrogen gas (N₂) or other gas, isconnected to valve assembly 2828 by fitting 2851. Valves 2840 and 2841regulate the flow of N₂ into their respective containers. In someembodiments, additional containers (not shown) may be provided, similarto container 2810, for introducing a buffer and/or other fluid into thecavity.

In operation, a fluid is placed into container 2810. The fluid, forexample, may contain targets that are to be hybridized with probes onthe chip. Container 2810 is sealed by closing port 2811 while container2820 is vented by opening port 2821. Next, N₂ is injected into container2810, forcing the fluid through tube 2850, cavity 310, and finally intocontainer 2820. The bubbles formed by the N₂ agitate the fluid as itcirculates through the system. When the amount of fluid in container2810 nears empty, the system reverses the flow of the fluid by closingvalve 2840 and port 2821 and opening valve 2841 and port 2811. Thiscycle is repeated until the reaction between the probes and targets iscompleted.

In some applications, foaming may occur when N₂ interacts with thefluid. Foaming potentially inhibits the flow of the fluid through thesystem. To alleviate this problem, a detergent such as CTAB may be addedto the fluid. In one embodiment, the amount of CTAB added is about 1millimolar. Additionally, the CTAB affects the probes and targetspositively by increasing the rate at which they bind, thus decreasingthe reaction time required.

The system described in FIG. 28 may be operated in an alternativemanner. According to this technique, back pressure formed in the secondcontainer is used to reverse the flow of the solution. In operation, thefluid is placed in container 2810 and both ports 2811 and 2821 areclosed. As N₂ is injected into container 2810, the fluid is forcedthrough tube 2850, cavity 310, and finally into container 2820. Becausethe vent port in container 2820 is closed, the pressure therein beginsto build as the volume of fluid and N₂ increases. When the amount offluid in container 2810 nears empty, the flow of N₂ into container 2810is terminated by closing valve 2840. Next, the circulatory system isvented by opening port 2811 of container 2810. As a result, the pressurein container 2820 forces the solution back through the system towardcontainer 2810. In one embodiment, the system is injected with N₂ forabout 0.3 seconds and vented for about 3 seconds. This cycle is repeateduntil hybridization between the probes and targets is completed.

FIG. 29 illustrates an alternative embodiment of the agitation system,System 2900 includes a vortexer 2910 on which the chip package 300 ismounted. A container 2930 for holding the fluid communicates with inlet350 via tube 2950. A valve 2935 may be provided to control the flow ofsolution into the cavity. In some embodiments, circulator 2901, whichmay be a N₂ source or other gas source, is connected to container 2930.Alternatively, a pump or other fluid transfer device may be employed.The flow of N₂ into container 2930 is regulated by a valve 2936.Circulator 2901 is also connected to inlet tube 2950 via a valve 2902.

A waste container 2920 communicates with port 360 via outlet tube 2955.In one embodiment, a liquid sensor 2940 may be provided for sensing thepresence of liquid in outlet tube 2955. Access to the waste containermay be controlled by a valve 2921. Optionally, additional containers(not shown), similar to container 2930, may be employed for introducinga buffer or other fluid into the cavity.

The system is initialized by closing all valves and filling container2930 with, for example, a fluid containing targets. Next, valves 2936,2935, and 2921 are opened. This allows N₂ to enter container 2930 whichforces the fluid to flow through tube 2950 and into the cavity. When thecavity is filled, valves 2935, 2936, and 2921 are closed to seal thefluid in the cavity. Next, the vortexer is activated to vibrate the chippackage, similar to a paint mixer. In some embodiments, the vortexer mayvibrate the package at about 3000 cycles per minute. The motion mixesthe targets in the fluid, shortening the incubation periods in someembodiments, the vortexer rotates the chip package until hybridizationis completed. Upon completion, valve 2902 and 2955 are opened to allowN₂ into the cavity. The N₂ empties the fluid into waste container 2920.Subsequently, the cavity may be filled with a buffer or other fluid.

FIG. 30 illustrates an alternative embodiment in which the agitationsystem is partially integrated into the chip package. As shown, chippackage 300 includes a cavity 310 on which the chip is mounted. Cavity310 is provided with inlets 360 and 350. The package also includeschambers 3010 and 3020. A port 3021 is provided in chamber 3010 and isconnected to inlet 360 by a channel 3025.

Chamber 3010 is equipped with ports 3011 and 3012. Port 3012communicates with inlet 350 through a channel 3015. Channel 3015 isprovided with a waste port 3016 that communicates with a fluid disposalsystem 3500 via a tube 3501. A valve 3502 regulates the flow of fluidsinto the disposal system. In some embodiments, the disposal systemincludes a waste container 3510 and fluid recovery container 3520 whichare connected to tube 3501. A valve 3530 is provided to direct the flowof fluids into either the waste container or recovery container.

Port 3011 is coupled to a fluid delivery system 3600 through a tube3601. Fluids flowing into chamber 3010 from the fluid delivery systemare regulated by a valve 3602. The fluid delivery system includes fluidcontainers 3610 and 3620 that are interconnected with a tube 3690.Container 3610, which may hold a fluid containing targets, includesports 3616 and 3615. Port 3616 is connected to tube 3690. A valve 3612controls the flow of the fluid out of container 3610. A circulator 3605,which may be a N₂ source, is connected to port 3615 of container 3610.Alternatively, any type of gas, pump or other fluid transfer device maybe employed. The flow of N₂ into container 3610 is controlled by a valve3618. A valve 3619 may also be provided to vent container 3610.

Container 3620, which may hold a buffer, is provided with ports 3625 and3626. Circulator 3605 is connected to port 3625. A valve 3621 isprovided to control the flow of N₂ into container 3620. Port 3626 isconnected to tube 3690 via a valve 3622. Valve 3622 regulates the flowof the buffer out of container 3620. Optionally, additional containers(not shown), similar to container 3620, may be configured forintroducing other fluids into the cavity. A valve 3690 connectscirculator 3605 to tube 3690 for controlling the flow of N₂ directlyinto the package. A valve 3652 is provided for venting the fluiddelivery system.

In the initial operating state, all valves are shut. To start thehybridization process, a fluid containing targets is introduced intochamber 3010 by opening valves 3602, 3612 and 3618. This injects N₂ intocontainer 3610 which forces the fluid to flow through 3601 and intochamber 3010. When chamber 3010 is filled, valves 3612 and 3618 areclosed. Next, valve 3642 is opened, allowing N₂ to flow directly intochamber 3010. The N₂ agitates and circulates the fluid into cavity 310and out to chamber 3020. As the volume of fluid and N₂ in chamber 3020increase, likewise does the pressure therein. When chamber 3020approaches its capacity, valve 3642 is closed to stop the fluid flow.Thereafter, the system is vented by opening valve 3652. Venting thesystem allows the back pressure in chamber 3020 to reverse the flow offluids back into chamber 3010. When chamber 3010 is filled, valve 3652is closed and valve 3642 is opened to reverse the fluid flow. This cycleis repeated until hybridization is completed.

When hybridization is completed, the system may be drained. Thisprocedure depends on which chamber the fluid is located in. If the fluidis located in chamber 3020, then valve 3502 is opened, while valve 3530is positioned to direct the fluid into the appropriate container(recovery or waste). The pressure in chamber 3020 forces the fluidthrough port 3016, tube 3501, and into the disposal system. If the fluidis in chamber 3010, then valve 3502 and 3642 are opened. As a result, N₂forces the fluid in chamber 3010 through port 3501 and into the disposalsystem.

Once the system is emptied, all valves are closed. A buffer or otherfluid may be introduced into the cavity. For example, the cavity may befilled with a buffer by opening valves 3602, 3621, and 3622. Thisinjects N₂ into container 3620 which forces the buffer therein to flowthrough the system until it fills cavity 310. In the alternative,ultrasonic radiation, heat, magnetic beads, or other agitationtechniques may be employed.

The present inventions provide commercially feasible devices forpackaging a probe chip. It is to be understood that the abovedescription is intended to be illustrative and not restrictive. Manyembodiments will be apparent to those skilled in the art upon reviewingthe above description. Merely as an example, the package may be moldedor machined from a single piece of material instead of two. Also, otherasymmetrical designs may be employed to orient the package onto thedetection systems.

The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

1. A method of making probe chips comprising the steps of: forming aplurality of probe arrays on a wafer; separating said wafer into aplurality of chips, each of said chips comprising at least one probearray thereon; and mating at least one of said chips to a package, saidpackage comprising a reaction chamber, said reaction chamber comprisinginlets for flowing fluid therein, said at least one probe array in fluidcommunication with said reaction chamber. 2-38. (canceled)